1. Field of the Invention
The present invention relates generally to the field of cancer treatment. More specifically, the present invention relates to the unexpected synergistic effects of nuclear transcription factor NF-κB inhibitors and anti-neoplastic agents in the treatment of cancer.
2. Description of the Related Art
Every year breast cancer is diagnosed in 910,000 women worldwide, and 376,000 women die from the disease (1). Most of these cases are in industrialized countries with 180,000 in North America and 220,000 in Europe. The highest reported rates of breast cancer incidence are for white or Hawaiian women in the United States and the rates in Africa and Asia are significantly lower (1). Immigrants from low-risk to high-risk regions experience rates of breast cancer incidence approaching those of the host country, suggesting lifestyle is the major contributor to the development of the disease. Only 5% of the cases have been assigned to inherited mutations in genes such as BRCA1 and BRCA2, while diet and environment may be responsible for as many as 50% of breast cancers (2).
Although the precise nature of the lifestyle risk factors causative for breast cancer are unknown, some of the known ones are age, race, ethnicity, hormones, and dietary factors (3, 4). Epidemiological and animal studies have shown that different microchemicals present in the diet could be effective agents for the prevention of cancer incidence and mortality (2, 5–6).
The induction of most cancers, for example breast cancer, is a multistep process initiated with DNA damage and followed by alteration of different signaling pathways. Usually, at the initial stages, breast cancer is hormone-dependent, relying on natural steroids. In later stages, however, growth of breast cancer becomes hormone-independent (7). Approximately 40% of the patients diagnosed with breast cancer have disease that has regional or distant metastases and, at present, there is no efficient curative therapy for breast cancer patients with advanced metastatic disease.
Paclitaxel (taxol), derived from the Pacific yew tree, is the first taxane used in clinical practice, and has shown a significant amount of anti-tumor activity in patients with breast cancer, ovarian cancer, head and neck cancer, non-small-cell lung cancer and sarcoma (8, 9). Taxanes represent a new class of antitumor agents that exert their action by promoting tubulin polymerization and microtubule assembly. Paclitaxel has been shown to induce in vitro apoptosis in various breast tumor cell lines and the degree of apoptosis inversely correlates with expression of HER2 in these cell lines (10, 11). How paclitaxel induces apoptosis is not fully understood (12), but a number of apoptosis-associated genes that either suppress, activate or mediate apoptosis are affected by paclitaxel. Tyrosine phosphorylation (13), microtubule assembly (14), bcl-2 phosphorylation (15), bcl-xl (16), p21, and p53 (17) have been implicated.
In addition to activating the apoptosis pathway, paclitaxel also simultaneously activates the anti-apoptotic pathway through induction of NF-κB in macrophages (18), ovarian cells (19), lung cancer cells (20), and breast tumor cells (21). The NF-κB activation by paclitaxel leads to the expression of various genes including interleukin-8 (19, 20), IL-1, and TNF (22). Expression of IL-8 can either enhance growth as in the case of melanoma (23), or promote angiogenesis as in the case of human lung carcinoma (24). Several groups have shown that NF-κB activation could lead to suppression of apoptosis (25–28). Paclitaxel-induced apoptosis in leukemia cells is suppressed by the activation of NFκB (28). NF-κB has been shown to directly activate the expression of bcl-xl (29), and bcl-xl over-expression has been shown to suppress paclitaxel-induced apoptosis. Paclitaxel is also known to activate JNK and AP-1, which could contribute to its anti-apoptotic pathway (30).
NF-κB plays an essential role in the development and progression of breast cancer. Animal studies suggest the presence of constitutively active NF-κB at an early stage during neoplastic transformation of mammary cells (31). NF-κB inhibits apoptosis in mouse mammary epithelia (32) and selective activation of NF-κB subunits have been found in human breast cancer cell lines and patient samples (33, 34). An inverse correlation between the levels of NF-κB activation and estrogen receptor expression has been reported (35) and inhibition of NF-κB in breast cancer cells induces spontaneous apoptosis (32, 34). Paclitaxel-induced sensitivity of breast cancer cell lines was enhanced by an NF-κB inhibitor, parthenolide (36, 37). The Mullerian inhibiting substance was also found to inhibit breast cancer growth through NF-κB mediated pathway (38). Furthermore, the transactivation function of NF-κB is negatively regulated by IκBβ1 in breast cancer cell lines (37). Lastly, overexpression of HER2/neu can activate NF-κB through the activation of Akt pathway and block apoptosis (39). All these reports together suggest that NF-κB may play an important role in breast cancer.
Curcumin (diferuloylmethane), a non-nutritive food chemical present in turmeric (Curcuma longa), has been found to be pharmacologically safe as indicated by consumption of curcumin as a dietary spice for centuries at doses up to 100 mg/day (40). Curcumin has been shown to block tumor initiation induced by benzo [a] pyrene and 7, 12 dimethylbenz [a] anthracene (41) and to suppress phorbol ester-induced tumor promotion (42, 43). Curcumin was found to suppress carcinogenesis of skin (43–47), forestomach (48, 49), colon (50–52), and liver (53) in mice. Curcumin has also been shown to suppress mammary carcinogenesis (54–56). Curcumin exhibits a number of characteristics that indicate curcumin would have potent chemopreventive activity. These characteristics are set forth below.
Curcumin Exhibits Antiproliferative Effects Against Tumor Cells
Curcumin has been shown to inhibit the proliferation of a wide variety of tumor cells including B cell and T cell leukemia (57–60), colon carcinoma (61), and epidermoid carcinoma (62). It has also been shown to suppress the proliferation of various breast carcinoma cell lines in culture (63–65). Growth of the breast tumor cell lines BT20, SKBR3, MCF-7, T47D, and ZR75-1 were completely inhibited by curcumin as indicated by MTT dye uptake, thymidine incorporation, and clonogenic assay (63). Curcumin can overcome adriamycin resistance in MCF-7 cells (63). Recently, curcumin was shown to activate caspase-8 which leads to cleavage of BID, thus resulting in sequential release of mitochondrial cytochrome C and activation of caspase-9 and caspase-3 (66).
Curcumin Downregulates the Activity of EGFR and Expression of HER2/neu
Effects on HER2/neu and EGFR may represent one possible mechanism by which curcumin suppresses the growth of breast cancer cells. Almost 30% of the breast cancer cases have been shown to overexpress the HER2/neu protooncogene (67), and both HER2 and EGF receptors stimulate proliferation of breast cancer cells. Overexpression of these two proteins correlates with progression of human breast cancer and poor patient prognosis (67). Curcumin has been shown to downregulate the activity of EGFR (62, 68) and HER2/neu (69) and to deplete the cells of HER2/neu protein (69). Additionally, curcumin can downregulate bcl-2 expression, which may contribute to its antiproliferative activity (70).
Curcumin Downregulates the Activation of Nuclear Factor-κB
Curcumin may also operate through NF-κB. NF-κB is a nuclear transcription factor required for the expression of genes involved in cell proliferation, cell invasion, metastasis, angiogenesis, and resistance to chemotherapy (71). This factor is activated in response to inflammatory stimuli, carcinogens, tumor promoters, and hypoxia which is frequently encountered in tumor cells (72). Activated NF-κB suppresses apoptosis in a wide variety of tumor cells (25–27), and it has been implicated in chemoresistance (25). Cells that overexpress NF-κB are resistant to paclitaxel-induced apoptosis (28). Furthermore, the constitutively active form of NF-κB has been reported in human breast cancer cell lines in culture (35), carcinogen-induced mouse mammary tumors (73), and biopsies from patients with breast cancer (33). Various tumor promoters, including phorbol ester, TNF and H2O2 activate NF-κB and that curcumin downregulates the activation (74). Subsequently, others showed that curcumin-induced downregulation of NF-κB is mediated through suppression of IκBα kinase activation (75, 76).
Curcumin Downregulates the Activation of AP-1 and c-jun Kinase
AP-1 is another transcription factor that has been closely linked with proliferation and transformation of tumor cells (77). The activation of AP-1 requires the phosphorylation of c-jun through activation of stress-activated kinase c-jun N-terminal kinase (JNK) (78). The activation of JNK is also involved in cellular transformation (79). Curcumin has been shown to inhibit the activation of AP-1 induced by tumor promoters (80) and JNK activation induced by carcinogens (81).
Curcumin Suppresses the Induction of Adhesion Molecules
The expression of various cell surface adhesion molecules such as ICAM-1, VCAM-1 and ELAM-1 on endothelial cells are absolutely critical for tumor metastasis (82). The expression of these molecules is in part regulated by nuclear factor NF-κB (83). Treatment of endothelial cells with curcumin blocks the cell surface expression of adhesion molecules and this accompanies the suppression of tumor cell adhesion to endothelial cells (84). Downregulation of these adhesion molecules is mediated through the downregulation of NF-κB activation (84).
Curcumin Downregulates Cox2 Expression
Overexpression of cyclooxygenase (COX)-2 has been shown to be associated with a wide variety of cancers, including colon (85), lung (86), and breast (87) cancers. The role of COX2 in suppression of apoptosis and tumor cell proliferation has been demonstrated (88). Furthermore, celebrex, a specific inhibitor of COX2, has been shown to suppress mammary carcinogenesis in animals (89). Several groups have shown that curcumin downregulates the expression of COX2 protein in different tumor cells (76, 90), most likely through the downregulation of NF-κB activation (76), which is needed for COX2 expression.
Curcumin Inhibits Angiogenesis
For most solid tumors, including breast cancer, angiogenesis (blood vessel formation) is essential for tumor growth and metastasis (91). The precise mechanism that leads to angiogenesis is not fully understood, but growth factors that cause proliferation of endothelial cells have been shown to play a critical role in this process. Curcumin has been shown to suppress the proliferation of human vascular endothelial cells in vitro (92) and abrogate FGF-2-induced angiogenic response in vivo (93), thus suggesting curcumin is also an antiangiogenic factor. Indeed curcumin has been shown to suppress angiogenesis in vivo (94).
Curcumin Suppresses the Expression of Matrix Metalloprotease (MMP)-9 and Inducible Nitric Oxide Oxidase (Inos)
MMP-9 is one of the proteases that has been shown to be regulated by NF-κB activation (96), and curcumin has been shown to suppress its expression (96). Curcumin has also been demonstrated to downregulate iNOS expression, also regulated by NF-κB and involved in tumor metastasis (97). All these observations suggest that curcumin must have anti-metastatic activity. Indeed, there is a report suggesting that curcumin can inhibit tumor metastasis (98).
Curcumin Downregulates Cyclin D1 Expression
Cyclin D1, a component subunit of cyclin-dependent kinase (Cdk)-4 and Cdk6, is rate limiting in progression of cells through the first gap (G1) phase of the cell cycle. Aberrant overexpression of cyclin D1 is associated with breast cancer formation, with cyclin D1 mRNA overexpressed in 70–100% of breast cancer cell lines and the majority of breast cancers (99). Targeted overexpression of cyclin D1 induced mammary adenocarcinoma (100), and transgenic mice lacking both cyclin D1 alleles failed to develop normal mammary glands (101). Furthermore cyclin D1 is required for transformation by activated HER2/neu (102). Antisense to cyclin D1 has been shown to induce apoptosis (103). Thus cyclin D1 downregulation has been suggested as one of the target for the treatment of breast cancer. Retinoic acid, a chemopreventive agent, has been shown to diminish cyclin D1 protein but not the mRNA through post-translational regulation (104). Expression of cyclin D1 is also regulated through NF-κB (105). Recent studies indicate that curcumin can rapidly downregulate the expression of cyclin D1 at the transcriptional and post-transcriptional level, and this may contribute to the antiproliferative effects of curcumin against various cell types.
The prior art is deficient in clinical strategies that augment the therapeutic efficacy of existing antineoplastic agents against cancer with a NF-κB-blocking chemopreventive agent. The present invention fulfills this long standing need and desire in the art by demonstrating the efficacy of a combination therapy approach involving an NF-κB blocker such as curcumin.